The Enterococcus secretome inhibits the growth of Mycobacterium tuberculosis complex mycobacteria

Enterococcus mundtii , a commensal intestinal bacterium, was demonstrated to inhibit the growth of some Mycobacterium tuberculosis complex (MTC) species that cause tuberculosis in humans and mammals. To further explore this preliminary observation, we cross-investigated five E. mundtii strains and seven MTC strains representative of four MTC species using a standardized quantitative agar well diffusion assay. All five E. mundtii strains, calibrated at 10 MacFarland, inhibited the growth of all M. tuberculosis strains with various susceptibility profiles, but no inhibition was observed with lower inoculums. Further, eight E. mundtii freeze-dried cell-free culture supernatants (CFCS) inhibited the growth of M. tuberculosis , Mycobacterium africanum, Mycobacterium bovis and Mycobacterium canettii, the most susceptible MTC species (inhibition diameter 25±1 mm), proportionally to CFCS protein concentrations. The data reported here indicate that the E. mundtii secretome inhibited growth of all MTC species of medical interest, which broadens previously reported data. In the gut, the E. mundtii secretome may modulate the expression of tuberculosis, exhibiting an anti-tuberculosis effect, with some protective roles in human and animal health.


INTRODUCTION
Tuberculosis is a deadly infection in mammals and humans, transmissible by the transcutaneous, digestive and especially respiratory tract routes, caused by any of the 13 closely related species of mycobacteria forming the Mycobacterium tuberculosis complex (MTC) [1]. Among these species, Mycobacterium tuberculosis, Mycobacterium bovis and its derivative, M. bovis Bacillus bilié de Calmette-Guérin (BCG), are the most frequently encountered species in clinical microbiology laboratories worldwide [2], and Mycobacterium africanum and Mycobacterium canettii is are mainly documented in patients exposed in the Horn of Africa [3,4]. Of particular concern are cases of tuberculosis caused by MTC isolates resistant to first-line rifampicin-based anti-tuberculosis combinations, stimulating efforts to evaluate new drugs, as well as the reintroduction of old drugs, such as those used for decades as anti-leprosy drugs [5,6].
Over the last decade, several lines of observation have indicated reciprocal interactions between the gut microbiota and MTC mycobacteria [7][8][9]. MTC mycobacteria can be found in intestinal tissues and faeces in the case of intestinal tuberculosis, and in the faeces in the case of pulmonary tuberculosis [10]. Accordingly, excreted faeces could be used as surrogate specimens on which to base the detection of M. tuberculosis by PCR and culture [11]. Experimental models incorporating either M. canettii or M. tuberculosis have confirmed lymphatic and pulmonary tuberculosis after an oral route of transmission of these MTC pathogens [12]. Finally, these experimental data agree with medical observations made during the so-called Lübeck disaster in Germany in 1929-1933, where 251 neonates were vaccinated by the oral route with a BCG vaccine contaminated with M. tuberculosis, 173 neonates developed tuberculosis and 72 died [13]. Increasingly, gut microbiota distortions are being implicated in the pathogenesis of several infectious and non-infectious diseases. Therefore, the role of gut microbiota in modulating the natural history of tuberculosis is being increasingly considered, yet is not entirely understood [14]. A few recent studies have disclosed OPEN ACCESS significant differences in gut microbiota composition between tuberculosis and non-tuberculosis patients: in tuberculosis patients, pathogenic species of Actinobacteria and Proteobacteria were significantly more abundant, while faeces collected from apparently healthy individuals were enriched in Bacteroidetes and Firmicutes [15,16].
Bacteria of the genus Enterococcus are among Firmicutes commensals of the gastrointestinal tract [17] known to produce antimicrobial peptides [18][19][20]. In fact, Enterococcus species produce a wide range of diverse antimicrobial peptides, often more than one per strain, some of which are atypical and distinct from known antimicrobial peptides [21]. Enterococci have received increased attention in recent years, due to their applications in medical treatments and their consideration as beneficial organisms for health [17,22].
Recently, we reported the in vitro inhibitory effect of Enterococcus mundtii against MTC species [23]. Here, we further investigated the experimental anti-tuberculosis activity of different Enterococcus species to characterize and explore the inhibition spectrum of the Enterococcus secretome against some MTC representative species.

Enterococcus strains
A total of 27 Enterococcus species were investigated in this study. These strains were grown on Columbia agar solid medium, supplemented with 5 % sheep blood (COS; bioMérieux) for 24 h in an aerobic atmosphere enriched with 5 % CO 2 at 37 °C. E. mundtii colonies were suspended in 1 ml of DPBS, vortexed for 2 min and adjusted to 10 McFarland equivalent of 3×10 9 c.f.u. ml -1 according to serial dilution methodology (Table 1).

E. mundtii cell-free culture supernatant
E. mundtii strains were grown in De Man, Rogosa, and Sharpe broth (MRS broth; Sigma-Aldrich) prepared as follows: 1 litre of MRS broth supplemented with 1 ml of Tween 80 (Sigma-Aldrich) was autoclaved at 121 °C for 15 min. A 500 µl volume of E. mundtii suspension inoculated into 100 ml of autoclaved MRS broth was incubated at 30 °C for 18 h under aerobic conditions. Then, E. mundtii cultures were centrifuged for 15 min at 3200 g at 4 °C and the supernatant was filtered using a 0.22 µm filter (Carl Roth) in order to obtain a cell-free culture supernatant (CFCS). The CFCS was adjusted to pH 6.5 using an NaOH solution to eliminate any pH-dependent bias, and 20 ml of CFCS was lyophilized for 24 h at −80 °C. Lyophilized CFCS was resuspended in 2 ml of DPBS, then diluted with DPBS at 1:2, 1:5, 1:8 and 1:10. CFCS dilutions were inoculated on COS medium for 24 h at 37 °C in an aerobic atmosphere enriched with 5 % CO 2 to ensure their sterility. To determine the nature of inhibitory factors, E. mundtii CFCS was incubated with proteinase K (1 mg ml −1 ) at 56 °C for 2 h, prior to inhibition testing as above. Also, heat resistance was assessed by incubating the E. mundtii CFCS for 15 min at 100 °C, prior to inhibition testing as above. Protein concentration was quantified by the Qubit assay (Bio-Rad). Non-inoculated MRS broth was used as a negative control throughout the experimental process.

Agar well diffusion assay
E. mundtii anti-tuberculosis activity was evaluated by the agar well diffusion assay. Initially, a mycobacterial suspension was inoculated on Middlebrook 7H10 agar for 24 h at 37 °C in an aerobic atmosphere enriched with 5 % CO 2 . Two wells were prepared in the centre of the 7H10 agar medium Petri dish: 100 µl of E. mundtii suspension was inoculated in a first well, and 100 µl of DPBS was inoculated in the other well as a negative control. Then, the anti-tuberculosis activity of E. mundtii CFCS was evaluated using 100 µl of CFCS in a first well, while 100 µl of MRS broth was used as a negative control in the second well. Petri dishes were incubated at 37 °C for 15 days under an aerobic condition enriched with 5 % CO 2 . The 1:2, 1:5, 1: and 1:10 CFCS dilutions were tested respectively and the diameter of MTC growth inhibition area around each well was measured in millimetres focusing restrictively on the inhibition areas. The E. mundtii CSURP2005 strain and its CFCS diluted at 1:5 were used to standardize the agar well diffusion assay conditions against other strains of MTC, including M. canettii, M. bovis and M. africanum; all manipulations were performed in triplicate (Fig. 1).

Statistical analyses
Data were entered into Microsoft Excel for Office to calculate the mean of inhibition diameters. Statistical analysis was done using GraphPad software (v8.0). A one-way ANOVA test was used to compare the mean of the inhibition diameters of E. mundtii strains and to compare the susceptibility of mycobacteria to enterococci. Differences were considered statistically significant at P<0.05.

E. mundtii suspension anti-tuberculosis activity
The agar well diffusion assay indicated that the five E. mundtii strains evaluated here inhibited the growth of all M. tuberculosis strains: a clear inhibition area was observed around the well containing the 10 MacFarland inoculum of E. mundtii suspension after a 15 day inoculation, but not with the 8 MacFarland or 5 MacFarland suspension, suggesting that the effect observed here was dependent on the E. mundtii inoculum in the absence of any growth inhibition zone around the DPBS negative control (Fig. 2a). Comparing the inhibition zone diameters resulting from cross-investigation of the five E. mundtii strains and the four M. tuberculosis strains (S10, S14, S15, S16) revealed that E. mundtii strains had a significantly different inhibitory effect as follows: E. mundtii CSURQ1712 and E. mundtii CSURP7988 between two strains M. tuberculosis S10 and M. tuberculosis S15 (P<0.05) ( Fig. 2bE and bF), E. mundtii CSURP724 between M. tuberculosis S10 and M. tuberculosis S14 (P<0. 05) (Fig. 2bG), E. mundtii CSURP5399 between M. tuberculosis S10 and all other M. tuberculosis strains tested (P<0.05) (Fig. 2bH), and finally   tuberculosis strain S10, M. tuberculosis strain S14, M. tuberculosis strain S15 and M. tuberculosis strain S16 to E. mundtii strains using one-way ANOVA test. Statistical significance was defined as *P<0.05.

CFCS anti-tuberculosis activity
The protein concentration in each CFCS dilution of five E. mundtii strains ( M. tuberculosis strain S10, the five E. mundtii strains, CFCS diluted to 1:2, 1:5 and 1:8, showed anti-tuberculosis activity, while no zone of inhibition was observed with the 1:10 diluted CFCS and the negative control in MRS broth (Fig. 3a). An area of inhibition was observed around the well containing the CFCS, which varied with CFCS concentration; the 1:8 dilution showed the lowest inhibition activity (inhibition diameter 9±1 mm). Dilution less than 1:8 showed no effect against M. tuberculosis strains (Table 4). A significant difference was observed between the three dilutions of five CFCS (P=0.001) (Fig. 3b). The inhibition diameters observed here were proportional to the CFCS fraction and the protein content of each dilution (Fig. 3c). The E. mundtii CSURP2005 strain exhibiting the highest protein content and its 1:5 dilution with a more representative inhibition zone was chosen to standardize the agar well diffusion assay used in this study. Treating E. mundtii CFCS with proteinase K resulted in total disappearance of the inhibition zones, suggesting the protein nature of the inhibitory factors. Furthermore, anti-tuberculosis activity was not affected by 15 min of heating at 100 °C. Also, from a total of 27 Enterococcus species, eight showed anti-tuberculosis activity against four MTC species. More precisely, 15 strains showed anti-tuberculosis inhibitory effects, including five E. mundtii strains (CSURQ1712, CSURP724, CSURP7988, CSURP5399 and CSURP2005), two E. avium strains (CSURQ4946 and CSURQ5145), two E. durans (CSURP8822 and CSURQ2511), one E. casseliflavus strain (CSURQ3866), one E. faecalis strain (CSURP6215), one E. faecium strain (CSURP3600), one E. hirae strain (CSURQ3681), one E. dispar strain (CSURP0234) and one E. devriesei strain (CSURP0494), with variable susceptibility between strains (Fig. 4). There was no significant difference in the susceptibility of M. tuberculosis, M. bovis and M. africanum, but M. canettii exhibited the highest susceptibility, with an inhibition area diameter of 25±1 mm (P=0.001) (Fig. 5).

DISCUSSION
The fact that E. mundtii inhibited the growth of some MTC species had been previously described in our laboratory [23], and the data reported here broadened such previous observations by cross-incorporating a set of E. mundtii strains and MTC species

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Peer review history
The conclusion statement needs to be modified. Rather than the work showing that Enterococci are likely to be key players in the control of Mycobacteria in the gut a more realistic conclusion might be that the results of this study suggest that further investigations are warranted to see if enterococci play any role in vivo in the control of Mycobacteria in the gut.

Authors 'answer:
The reviewer is perfectly right and the conclusion statement has been corrected accordingly (lines 249-251).
Furthermore, the findings of the present study support the need for additional research to investigate the potential contribution of Enterococcusto the control of mycobacteria.

Comment 5:
Presentation of the results could be improved in places. Labelling of some of the figures needs attention.   Authors 'answer:Corrected accordingly.

Comment 1:
The manuscript by Achache et al describes experiments which tested the secretome of Enterococci on Mycobacterium tuberculosiscomplex mycobacteria. The methodology involved culture supernatants in agar diffusion assays and measurement of the zones of clearance. I was excited to read this manuscript as the topic is interesting and the research questions timely and important. However, I encourage the authors to develop their manuscript for clarity and robustness of data.
Authors 'answer:The authors acknowledge this enthusiastic, positive general comment.

Comment 2:
It is not clear to me that the experiments reported have been replicated as would be the standard in biology and a requirement for good statistics. Have these experiments been performed multiple independent times? Could the authors provide details? If replicates have been performed, could the data for these replicates be included Authors 'answer:Indeed, every single experiment was triplicate, as clarified in the M & M section in line 158. The mean of the 3 measurements (triplicate) are provided in Table 2 and Table 3.

Comment 3:
Overall, I found the manuscript confusing to read. Perhaps treating the Enterococcusand Mycobacteriumseparately and giving them separate methods sections might help? Also see comments below on Figure one.
Authors 'answer:The authors followed this suggestion: Mycobacteriumstrains (list, culture and confirmatory identification) are now given a separate section (lines 106-120); Entorococcusstrains (list, culture and confirmatory identification) are now given a separate section (lines 121-126), and the following sections remained unchanged.

Comment 4:
Many of the diffusion assays presented show zones of clearance that breach the agar plate and I don't believe these can be used to measure inhibition zones reliably. These agar plate might inform the authors, but the formal analysis should only relate to complete zones of clearance.
Authors 'answer:The perfectly is perfectly right and in this situation indeed the authors tabulated the measurable (smaller) diameter. This point is now clarified in the M & M section, line 155.

Comment 5:
Statistics: Can the authors confirm the number of independent replicates? Can the authors discuss their statistical approach? It is not clear to me what value a one-way ANOVA brings to this story, without multiple comparisons within the ANOVA.
Authors 'answer:The experiment was performed once with three agar plates for each test. The inhibition diameters were measured for each agar plate and an average of the inhibition diameters was calculated. The analysis is based on a single variance (one way ) that targets a single factor, M. tuberculosisstrainssusceptible or not.

Comment 6:
Line 87 all strains were identified … if they authors wish to include this statement, can they also include the data or reference if Comment 16: Authors 'answer:Done accordingly.
2. Remove the shadow from behind the agar plate picture. Also, the blue box outline is unnecessary and could be removed.
3. Have the ANOVA tests been performed to test significance of individual strain combinations? Could the authors explain the logic of performing an ANOVA on this group of strains (S10, S14, S15, S16) without making individual comparisons. This can easily be done in Graphpad and would add value to these data. For example, I suspect S15 treated individually might be significantly different from S14 (if only slightly and probably not meaningfully) in the CSURQ1712 graph.
4. Each graph should be its own panel and therefore have an identifying letter. E.g. Figure   1. It is not advisable to have figure 3, panel a sub-panel a,… please relabel these sub panels (i, ii… etc) or make each plate it's own panel.
2. Panel A (a) here an asymmetric diffusion pattern is shown. Ideally, this assay should be performed with either an agar plate large enough or a well small enough to allow a symmetric and entire diffusion pattern occur. Could the authors please explain why this hasn't been possible? I believe the 1/8 dilution shown in (c) is sufficient to measure the zone of clearance, but the authors have indicated they used the 1/5 which also doesn't show a full diffusion pattern.
Authors 'answer:The perfectly is perfectly right, in this condition, asymmetric diffusion patterns may be due to the concentration of the supernatant and how it is deposited and diffused onto the agar plate.
3. B I believe this graph would benefit from larger data points and perhaps a grey error bar.
Authors 'answer:Done accordingly. 4. I have concerns about how the data for dilution 1/2 and 1/5 were recorded in light of the images presented in this figure. It appears only the 1/5 dilution has error bars, but I would expect error in these types of data. Please clarify.
Authors 'answer:The graph has been modified so that the error bars are visible. 5. C The legend indicates a 1/10 dilution that is not present on the graph. It is difficult to see which bars correspond to the patterns shown in the legend. Perhaps increasing the bar size would help. Are there again no errors bars from these data? If not, please explain.

Authors 'answer:
The values for the 1/10 dilution are nul and were removed from the graph.  Remove all shadows from boxes Authors 'answer:Done accordingly.
The panel labels (A, B…) are not aligned and make the figure difficult to interpret. These panel labels could also benefit from being bigger. I would again strongly advise the red/green colour scheme and encourage a colour-blind friendly scheme. The legend for the red and green colours is poorly placed and closer to the strain names than the colour label.

Figure 5
This figure is missing panel labels, while the wells seem to be labelled C and D.
I have concerns about how this diffusion was measured given that the agar plate is too small to measure the full zone of clearance.
Authors 'answer:Done accordingly. See above answer. Table 1 Each cell showing a measurement in the table should be the same size and appear uniformly in this table.
As the authors did answer all the reviewers 'remarks and queries, did perform additional experiments as required, and revised the manuscript accordingly, they hope that this revised version will be accepted for publication.
to include this statement, can they also include the data or reference if this has already been published. Suggestion: the extensive list of strains might be better placed in a table than text. This might aid readability. Line 126 the abbreviation DPBS is explained here but has already been cited at an earlier line without explanation. Please define abbreviation at the first use. Line 137 could the authors please clarify why cell free culture supernatant was pH adjusted? Line 141 could the authors please indicate why 1/5 dilution was chosen? I find the descriptions of CFU/ mL and/or MacFarland standars equivalants difficult to follow when their use is interchangeable in the text. Could the authors please use both or just CFU / mL alone. Line 173 -177 I find this sentence confusing. The texts describes a similar inhibitory effect of CSURQ1712 and CSURP7988 but cites p values of 0.1 and 0.2. These p values do not indicate significant differences. Could the authors please correct this. I suspect there is either a typo here, or the p value doesn't relate to the inhibition zones shown. This section required clarification. Line 187 The protein values cited in the text should have standard deviations. Multiple mentions of standardisation of the well diffusion assay to the 1/5 dilution. The methods would benefit from detailing exactly what this standardisation is and how its performed. Line 218 "negativity of negative controls" Rephrasing this to "absence of zones of clearance around the negative controls" or similar scientific language is recommended. Line 219 The inhibitory effect was proportional to the inoculum… but this contradicts line X which states an effect was only observed at Y. I understood the statement in line X to indicate the effect was an on/off type effect. Please clarify these statements for accuracy. Line 231 "at large" suggestion to remove "at large" Line 257 is "validation of manuscript" correct here? It is surprising that the protein concentration differs so little between the 1/8 dilution and the 1/10 dilution, however the zone of clearance effect is completely lost. Could the authors speculate or discuss this in the discussion? Figure 1 I commend the authors for including a schematic figure describing their methodology and I would strongly encourage the authors to improve this figure. I have the following suggestions: 1.
remove all shadows from boxes and pictures 2. revise colour scheme from a red/green to a colour-blind friendly pallet 3. slightly reduce the size of the agar plates at the bottom of the figure 4. use the exact same agar plate diagram and size for each plate -change the colour to indicate media and insert circles to indicate well. 5. Make all test tubes the same size and if you must have them at an angle, use the exact same angle for each test tube. 6. Make arrows uniform in size and colour 7. Make text uniform -don't present text in a coloured box or circle and remove coloured boxes. 8.
The figure required further explanation in the legend -what are 1, 2 and 3 in the schematic referring to? Figure 2 1. Perhaps the deviation bars or data points could be slightly different colours or thicknesses to aid visualisation? Currently, it is difficult to see individual data points that sit on the error bars. Perhaps grey error bars would help? 2. Remove the shadow from behind the agar plate picture. Also, the blue box outline is unnecessary and could be removed. 3. Have the ANOVA tests been performed to test significance of individual strain combinations? Could the authors explain the logic of performing an ANOVA on this group of strains (S10, S14, S15, S16) without making individual comparisons. This can easily be done in Graphpad and would add value to these data. For example, I suspect S15 treated individually might be significantly different from S14 (if only slightly and probably not meaningfully) in the CSURQ1712 graph. 4. Each graph should be it's own panel and therefore have an identifying letter. E.g. Figure 2 B-F. 5.
Line 426 the first description of this strain being obtained from human stool should not be in the figure legend. Please remove this detail from the legend and document this in the methods. Figure 3 1.
It is not advisable to have figure 3, panel a sub-panel a,… please relabel these sub panels (i, ii… etc) or make each plate it's own panel. 2.
Panel A (a) here an asymmetric diffusion pattern is shown. Ideally, this assay should be performed with either an agar plate large enough or a well small enough to allow a symmetric and entire diffusion pattern occur. Could the authors please explain why this hasn't been possible? I believe the 1/8 dilution shown in (c) is sufficient to measure the zone of clearance, but the authors have indicated they used the 1/5 which also doesn't show a full diffusion pattern. 3. B I believe this graph would benefit from larger data points and perhaps a grey error bar. 4.
I have concerns about how the data for dilution 1/2 and 1/5 were recorded in light of the images presented in this figure. It appears only the 1/5 dilution has error bars, but I would expect error in these types of data. Please clarify. 5.
C The legend indicates a 1/10 dilution that is not present on the graph. It is difficult to see which bars correspond to the patterns shown in the legend. Perhaps increasing the bar size would help. Are there again no errors bars from these data? If not, please explain. 6.
Please remove the blue boxes surrounding the figure. Figure 4 Remove all shadows from boxes The panel labels (A, B…) are not aligned and make the figure difficult to interpret. These panel labels could also benefit from being bigger. I would again strongly advise the red/green colour scheme and encourage a colour-blind friendly scheme. The legend for the red and green colours is poorly placed and closer to the strain names than the colour label. Figure 5 This figure is missing panel labels, while the wells seem to be labelled C and D. I have concerns about how this diffusion was measured given that the agar plate is too small to measure the full zone of clearance. Table 1 Each cell showing a measurement in the table should be the same size and appear uniformly in this table.

Please rate the manuscript for methodological rigour Poor
Please rate the quality of the presentation and structure of the manuscript Poor To what extent are the conclusions supported by the data? Partially support